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Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.
J Cell Biol 1989 Dec
PMID:Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. 259 99

We have cloned and sequenced the nitrate reductase (NR)-encoding gene (nia) from tomato. When compared to the two Nicotiana tabacum nia structural genes, this 5-kb tomato gene shows a highly conserved structure, the coding sequence being interspersed with three introns at the same positions. Nucleotide sequences of the 5' promoter regions are not homologous, except for a 250-bp fragment. This small region might be involved in the similar regulation of the nia expression in tomato and tobacco plant species. The tomato gene codes for a 911 amino acid (aa) polypeptide chain. This sequence was aligned with and compared to other higher plant NR sequences. This alignment clearly identifies the three catalytic domains of NR, namely, a molybdopterin cofactor-binding domain, a heme domain and a FAD/NADH domain. On the other hand, it suggests that the less conserved 80-aa N-terminal region, containing a striking acidic aa cluster, is an additional domain bearing regulatory or structural function.
Gene 1989 Dec 28
PMID:Cloning and analysis of the tomato nitrate reductase-encoding gene: protein domain structure and amino acid homologies in higher plants. 262 74

The amino acid sequence of the molybdenum-containing domain of chicken hepatic sulfite oxidase has been determined by Edman degradation of the purified protein. Combining these data with those previously published for the heme-containing domain (Guiard, B., and Lederer, F. (1979) Eur. J. Biochem. 100, 441-453) indicates that each subunit of the homodimer comprises a single polypeptide chain containing 460 amino acid residues (Mr = 50,545). Comparison of the sequence with the cDNA-deduced sequence of assimilatory nitrate reductase from Arabidopsis thaliana shows a substantial degree of sequence conservation in the regions of the proteins that have been identified as comprising the Mo-pterin- and cytochrome b557-binding domains. These results suggest that the sequences forming the molybdenum-binding domains of the molybdenum hydroxylases may have evolved from a common ancestral gene.
J Biol Chem 1989 Dec 15
PMID:Conserved domains in molybdenum hydroxylases. The amino acid sequence of chicken hepatic sulfite oxidase. 268 65

Hydrogenase activity and other hydrogenase-related functions can be restored to hydC mutants by the specific addition of nickel salts to the growth medium. These mutants are defective in all three hydrogenase isoenzymes and the restoration is dependent upon protein synthesis. The cellular nickel content of the mutant when grown in LB medium is less than 1% of that of the parental strain. Partial suppression of the hydrogenase phenotype of hydC mutants occurs when growth takes place in a different medium. This correlates with an increased cellular nickel content. The phenotype of the mutant is also fully suppressed by growth in media of very low magnesium content. Such media facilitate nickel uptake via the magnesium transport system, which leads to the acquisition of a normal cellular nickel content. Mutations in the fnr gene, which encodes a transcriptional regulator for several anaerobically expressed enzymes, abolishes hydC expression and gives rise to a defective hydrogenase phenotype. The hydrogenase phenotype of fnr is closely similar to that of hydC in all respects examined. The hydrogenase activity of fnr strains can be restored by the presence of a functional hydC gene on a multicopy plasmid. The hydrogenase phenotype of fnr strains therefore arises indirectly via suppression of hydC, which leads to a low cellular nickel content. Nickel has no influence on fumarate reductase or nitrate reductase activities in fnr strains. The hydrogen-metabolism phenotype of fnr strains is, therefore, dependent upon their ability to acquire nickel from growth media. It is likely that hydC encodes a specific transport system for nickel.
Mol Microbiol 1989 Dec
PMID:Nickel deficiency gives rise to the defective hydrogenase phenotype of hydC and fnr mutants in Escherichia coli. 269 44

Six Gram-negative bacteria (Klebsiella pneumoniae, Erwinia chrysanthemi, Proteus vulgaris, Serratia marescens, Salmonella typhimurium, and Pseudomonas aeruginosa) were shown to contain an FA-type protein capable of activating aponitrate reductase, apotrimethylamine N-oxide reductase and apoformate dehydrogenase of Escherichia coli. Protein FA activity was highest in Erwinia chrysanthemi and lowest in Pseudomonas aeruginosa. All the species also contained the low-Mr (less than or equal to 1500) heat-resistant material previously reported to be necessary for the protein-FA-dependent activation of E. coli chlB nitrate reductase.
J Gen Microbiol 1989 Dec
PMID:Escherichia coli molybdoenzymes can be activated by protein FA from several gram-negative bacteria. 269 94

The nit-3 gene of Neurospora crassa encodes the enzyme nitrate reductase and is regulated by nitrogen catabolite repression and by specific induction with nitrate. The nit-3 gene was isolated from a cosmid-based genomic library by dual selection for benomyl resistance and for the ability to complement a nit-3 mutant strain using the sibling-selection procedure. The nit-3 gene was subcloned as a 3.8-kilobase DNA fragment from a cosmid that carried an approximately 40-kilobase N. crassa DNA insert. A restriction fragment length polymorphism analysis revealed that the cloned segment displayed tight linkage to two linkage-group-4 markers that flank the genomic location of nit-3. RNA gel blot analyses of RNA from wild-type and various mutant strains were carried out to examine the molecular mechanism for regulation of nit-3 gene expression. The nit-3 gene was transcribed to give a large mRNA of approximately 3.4 kilobases, the expected size to encode nitrate reductase. The nit-3 gene was only expressed in wild-type cells subject to simultaneous nitrogen derepression and nitrate induction. A mutant of nit-2, the major nitrogen regulatory gene of Neurospora, did not have detectable levels of nit-3 gene transcripts under the exact conditions in which these transcripts were highly expressed in wild type. Similarly, a mutant of nit-4, which defines a minor positive-acting nitrogen control gene, failed to express detectable levels of the nit-3 transcript. Nitrate reductase gene expression was partially resistant to nitrogen repression in a mutant of the nmr gene, which appears to be a regulatory gene with a direct role in nitrogen catabolite repression. Results are presented that suggest that the enzyme glutamine synthetase does not serve any regulatory role in controlling nitrate reductase gene expression.
Proc Natl Acad Sci U S A 1987 Dec
PMID:Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in Neurospora crassa. 289 Nov 38

Escherichia coli K12 mutants lacking phenazine-methosulphate-linked formate dehydrogenase (FDH-PMS) activity, but still capable of producing normal levels of benzyl-viologen-linked formate dehydrogenase (FDH-BV) and nitrate reductase activities, have been isolated following P1 localized mutagenesis. The relevant mutations mapped with the same cotransduction frequency close to the rhaD gene, at 88 min on the E. coli chromosome. They were further subdivided into two classes. Class I consisted of six fdhD mutants which synthesized an inactive FDH-PMS protein with the same subunit composition as the wild-type enzyme. In contrast, class II contained four fdhE mutants totally devoid of this antigen. Construction of merodiploid strains harbouring various combinations of the mutated alleles, fdhE on the episome and fdhD on the chromosome, led to the restoration of FDH-PMS activity by complementation of the products encoded by the respective wild-type alleles. Difference spectroscopy suggested that both fdhD and fdhE mutants contained normal amounts of the cytochrome b559 associated with FDH-PMS although the cytochrome had lost its capacity for formate-dependent reduction.
J Gen Microbiol 1988 Dec
PMID:Mutants of Escherichia coli specifically deficient in respiratory formate dehydrogenase activity. 307 34

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.
J Biol Chem 1988 Dec 25
PMID:Limited proteolysis of the nitrate reductase from spinach leaves. 319 46

Ultraviolet light was shown to inactivate purified nitrate reductase in the presence of reduced benzyl viologen. Loss of activity was not complete, reaching 60 to 70%. Photolysis was maximum at 345 nm. The differential spectrum between native and irradiated enzyme exhibited absorption bands at 216, 275, 314 and 365 nm. The photosensitive electron carrier could be extracted by organic solvents. It had the following absorption bands: 225, 275 and 285 nm. It was reduced by Nile blue A but not by methylene blue. The precise nature of this light sensitive molecule could not be determined although the results support the idea that this chromophore might be a naphthoquinone.
Biochem Int 1987 Dec
PMID:An ultraviolet light sensitive target in nitrate reductase of Escherichia coli K-12. 332 2

Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the enzyme via hydrophobic forces as well as via an oxygen-sensitive mechanism. Upon extraction, the capability to complement the apo nitrate reductase of Neurospora crassa nit-1 can be conserved only in the total absence of oxygen. Cysteine and glutathione were shown to protect efficiently free mocofactor from oxidation. Two species of active mocofactor, probably a molybdoform and a demolybdoform, could be separated by means of reversed-phase HPLC with a mobile phase of 5 mM sodium citrate at a pH of 6.5. The mode of interaction between either of these species with thiol reagents is discussed.
Eur J Biochem 1987 Dec 01
PMID:Extraction and purification of molybdenum cofactor from milk xanthine oxidase. 369 96


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